Exposure of VWF thrombi to extracellular calcium induces spreading in tethering neutrophils. (A,B) Citrated whole blood was perfused over a VWF (100 μg/mL) matrix for 5 minutes at 1800 s⁻¹ to obtain platelet thrombi. Nonadherent platelets were removed by a further 5-minute perfusion with cell-free unsupplemented Tyrode buffer (VWF − calcium) or buffer supplemented with at 1800 s⁻¹ with 1 mM CaCl₂/MgCl₂ (VWF + calcium) for 5 minutes prior to perfusion of isolated neutrophils (1 × 10⁶/mL) resuspended in 1 mM CaCl₂/MgCl₂-containing Tyrode buffer for 5 minutes at 150 s⁻¹. Neutrophil-platelet interactions were visualized by phase contrast microscopy and the number of neutrophils exhibiting spreading (A) and stationary adhesion (B) quantitated. Results are presented as means (± SEM; n = 4). *P < .05. (C) Citrated whole blood was perfused over collagen (2 mg/mL) in the presence of FITC–annexin 5A (5 μg/mL) (green) and neutrophils were stained with DiIC₁₈ (1 μM) (orange) prior to perfusion as described in “Materials and methods, Perfusion studies, Platelet thrombi studies.” Platelets and neutrophils were simultaneously visualized using filters for DIC, and green and red fluorescence, by confocal microscopy. Image overlay is from 1 experiment representative of 4 independent experiments. Bar represents 30 μm.